The role of seminal plasma in the function, transport and fertility of ram spermatozoa

Transcription

1 The role of seminl plsm in the function, trnsport nd fertility of rm spermtozo T. Pini, J.P. Rickrd, G. Evns, W.M.C. Mxwell, S.P. de Grf Fculty of Veterinry Science, The University of Sydney, NSW 2006, Austrli Abstrct. To dte, there hve been no studies investigting the role seminl plsm plys in cervicl trnsit of epididyml rm spermtozo. As such, epididyml spermtozo were ssessed in the presence nd bsence of seminl plsm both in vitro nd in vivo. Experiment 1 exmined the effect of seminl plsm on fresh epididyml spermtozo with nd without subsequent cryopreservtion, mesuring motility vribles nd the bility to penetrte cervicl mucus. Motility prmeters of fresh epididyml spermtozo did not improve with exposure to seminl plsm. Only the totl motility of cryopreserved epididyml spermtozo significntly (p<0.001) improved with pre-freeze exposure to seminl plsm (verge exposed 31.9% ± 4.9% vs unexposed 20.4% ± 4%). However, dding seminl plsm significntly improved mucus penetrtion by fresh nd cryopreserved epididyml rm spermtozo (p<0.05). Experiment 2 investigted the fertility of epididyml spermtozo with nd without exposure to seminl plsm fter cervicl nd intruterine insemintion. While epididyml spermtozo performed poorly when inseminted cerviclly without seminl plsm (7.3%), exposure to seminl plsm yielded significntly (p=0.05) higher pregnncy rtes (37.0%). Tretment hd no significnt effect on pregnncy rtes following intruterine insemintion. These results suggest tht exposure to seminl plsm during ejcultion is necessry for norml survivl nd trnsit of spermtozo through the cervix. Additionl keywords: epididyml spermtozo, cervix, cervicl mucus, sheep Introduction Seminl plsm is the complex fluid secreted by the mjor ccessory sex glnds nd while it hs mny proposed functions, its exct role in reproduction remins uncler. Epididyml spermtozo, which hve never before hd contct with seminl plsm from the mjor ccessory sex glnds, hve been shown to hve surprisingly high fertility. This hs been demonstrted both in vitro through techniques such s intrcytoplsmic sperm injection nd IVF (Silber et l., 1995; Stout et l., 2012) nd in vivo when inseminted directly into the uterus (Fournier-Delpech et l., 1977; Hori et l., 2004; Hori et l., 2005; Ehling et l., 2006; Monteiro et l., 2011), in mny cses chieving pregnncy rtes similr to 1

2 those of uterine inseminted ejculted spermtozo (Fournier-Delpech et l., 1977; Ehling et l., 2006; Monteiro et l., 2011). Norml fertility of spermtozo which hve never come into contct with seminl plsm rises the question of whether exposure to seminl plsm from the ccessory sex glnds is biologicl requirement for norml sperm function in vivo or whether this substnce is lrgely superfluous to reproductive success. Despite these observtions, evidence lso exists tht seminl plsm plys beneficil role in reproductive processes nd fertility. It hs been demonstrted tht ddition of seminl plsm post thw cn improve the in vitro fertilising bility of cryopreserved ejculted rm spermtozo (El-Hjj Ghoui et l., 2007). Furthermore, it hs lso been suggested tht seminl plsm my improve the bility of cryopreserved ejculted rm spermtozo to nvigte the tortuous ovine cervix in vivo (Mxwell et l., 1999). While not definitive, this fosters the ide tht one of the min roles of seminl plsm could be ssisting cervicl migrtion of spermtozo. The cervicl migrtion model in sheep offers novel mens of testing the effect of seminl plsm on the bility of spermtozo to interct with the femle reproductive trct, s it is well estblished s site of high sperm selectivity (see Drurt, 2012 for review). No studies hve yet reported the fertility of epididyml spermtozo in the presence nd bsence of seminl plsm fter cervicl insemintion in the ewe. In ddition, while beneficil effects of seminl plsm hve been demonstrted in vitro using wshed, ejculted rm spermtozo, they hve yet to be replicted using epididyml spermtozo. As such, the im of the current study ws to exmine the effect of seminl plsm on the in vitro function of epididyml spermtozo with nd without subsequent cryopreservtion nd its influence on epididyml spermtozo trnsport nd fertility in vivo. We hypothesise tht exposure to seminl plsm will hve beneficil effects on importnt functionl chrcteristics nd in vivo fertility. Mterils & Methods Equipment nd regents All chemicls used were lbortory grde nd mnufctured by Sigm-Aldrich (Cstle Hill, Austrli) unless otherwise stted. Eppendorfs nd pipette tips were sourced from Eppendorf South Pcific Pty. Ltd (North Ryde, Austrli). Ultr-het treted (UHT) milk ws obtined from retil sources on the dy of ssessment. All experiments were ssessed nd pproved by the University of Sydney Animl Ethics Committee

3 Experiment 1: The effect of seminl plsm on the in vitro function of epididyml spermtozo, with nd without subsequent cryopreservtion Collection of ejculted semen nd seminl plsm Semen ws collected from mture merino rms (N=3) housed t the Gunn Building, University of Sydney, Cmperdown vi rtificil vgin in the presence of teser ewe. Ejcultes were immeditely ssessed for colour, volume nd wve motion (dt not shown). After ssessment, smples were diluted 1:1 with Slmon s Sheth Fluid (SSF; tris-citric cid, fructose) nd concentrtion determined using hemocytometer s described by Evns nd Mxwell (1987). Collections were subsequently eqully divided between fresh (F-EJAC) nd cryopreserved (C-EJAC) tretments. Seminl plsm ws obtined from severl ejcultes (collected during the breeding seson in 2013) of the sme three rms by centrifuging t 4000 g, once for 20 minutes, with the superntnt then collected nd spun for further 30 minutes t 4000 g (Sigm-Aldrich, Cstle Hill, Austrli). Seminl plsm smples were then seprted into liquots nd stored t -80 C. Individul liquots were thwed on ice s needed. Collection of epididyml spermtozo Rm testes (N=9 rms) were obtined t slughter from Goulburn bttoir nd trnsported on ice to the University of Sydney, Cmperdown where they remined chilled t 5 C for 24 hours. Epididyml spermtozo were obtined by microperfusion (Dcheux, 1980) using SSF. Collections were ssessed for wve motion (dt not shown) nd the concentrtion determined using hemocytometer s described by Evns nd Mxwell (1987). Epididyml collections were then hlved, with one hlf being undiluted epididyml spermtozo (EP) nd the reminder undergoing 1:1 dilution with seminl plsm previously collected from n nlogous rm (EPSP). Tretments were subsequently subjectively ssessed for motility (dt not shown). These two tretments were gin hlved, with one hlf remining fresh (F-EP, F-EPSP), nd the reminder being subsequently cryopreserved (C-EP, C-EPSP). Fresh smple preprtion Fresh tretments were diluted with UHT milk to stock solutions of spermtozo/ml, which were kept in 37 C wter bth over period of 6 hours. At the time of collection (0 hours), 3 hours nd 6 hours fter collection, 500µL liquot of ech smple ws diluted with Androhep (Minitube, Bllrt, Austrli) to spermtozo/ml for ssessment. 3

4 Cryopreserved smple preprtion Cryopreserved tretments were diluted to spermtozo/ml with Slmon s cryodiluent (tris-citric cid, glucose, egg yolk). Smples were chilled to 5 C over period of 2 hours nd liquots tken for pre-freeze ssessment. Chilled smples were subsequently loded into precooled strws (0.25mL; IMV Technologies, Germny), which were suspended bove liquid nitrogen for 8 minutes before being submerged. Strws were then stored in liquid nitrogen until use. Strws were thwed by gitting in 37 C wter bth for 2 minutes. A 500µL liquot of ech smple ws diluted with Androhep to spermtozo/ml t 0, 3 nd 6 hours post thw for ssessment. Motility A 5.5µL liquot of spermtozo/ml smple ws ssessed for motility prmeters by Computer Assisted Sperm Anlysis (CASA; IVOS II Animl, Hmilton Thorne, Beverly, USA), using CELL-VU slides (Millennium Sciences, Mulgrve, Austrli; pre-wrmed to 37 C) with 22 22mm cover slip (chmber depth 20µm). Motility prmeters were determined on n verge of t lest three rndom fields ( cells per smple) using fctory settings for rm, with smpling frequency of 60 Hz. Recorded vribles included motility, progressive motility, verge pth, curviliner nd stright line velocities, mplitude of lterl hed displcement, bet cross frequency, linerity nd strightness. Cervicl migrtion test Nturl cervicl mucus ws collected from synchronised merino ewes in oestrus, seprted into 500µL liquots nd stored t -80 C. Individul liquots were thwed on ice s needed. A vil of DNA-specific stin (IDENT; Hmilton Thorne, Beverly, USA) ws diluted with 500µL of UHT milk for fresh smples or Slmon s cryodiluent for cryopreserved smples, giving stock solution of 80µg/μL. Spermtozo were stined 1:1 with IDENT (finl working concentrtion 40µg/μL). Smples were incubted for 10 minutes then trnsferred to polyethylene cpsule (BEEM; ProSciTech, Thuringw, Austrli). A glss cpillry tube ( mm; Microslides, Mountin Lkes, USA) filled with nturl cervicl mucus nd seled with Cristsel (Hwksley, London, UK) ws immersed in the stined smple nd co-incubted (37 C, 1 h). Following incubtion, cpillry tubes were red under fluorescent microscopy (200 ; Olympus BX51) nd the number of spermtozo t 1cm nd the vngurd distnce (furthest spermtozoon) recorded

5 Experiment 2: The effect of seminl plsm on the in vivo fertility of fresh epididyml spermtozo Ewe synchronistion Oestrus ws synchronised in mture merino ewes (N=303), using combintion of intrvginl progesterone pessries (30mg; Ov-Gest; Bioniche, Armidle, Austrli) for 12 dys, followed by 400 IU of intrmusculr PMSG (1mL; Pregnecol; Bioniche, Armidle, Austrli) t sponge removl. Testosterone supplemented wethers (400mg dministered t sponge insertion nd further 150mg t removl; Durmte; Intervet, Austrli) were introduced to the flock t sponge removl, t rtio of 1 wether: 25 ewes. Collection of ejculted semen Ejcultes of mture merino rms (N=3; F-EJAC) were collected vi rtificil vgin immeditely prior to insemintion nd diluted 1:2 with wrmed UHT milk. Diluted smples were trnsported short distnce to the insemintion site, where they were ssessed for concentrtion nd motility (dt not shown). Smples were kept t 30 C prior to further dilution with UHT milk for insemintion. Collection of epididyml spermtozo Testes were removed from culled Merino rms (N=3 rms) nd epididyml spermtozo collected into sterile petri dish vi microperfusion with SSF (Dcheux, 1980). Following collection, epididyml spermtozo were centrifuged t 800 g for 10 minutes to concentrte the smple nd remove contminnts. The pellet ws subsequently resuspended in SSF. Epididyml tretments (F-EP, F-EPSP) were subsequently ssessed nd prepred s per experiment 1. Tretments were kept t 30 C prior to further dilution with UHT milk for insemintion. Insemintion Insemintions occurred over two dys in April 2013 t the University of Sydney property Arthursleigh in the New South Wles southern highlnds, with ll nimls held on site. In order to minimise niml stress nd mximise insemintion success, no working dogs were used nd ewes were given time to settle before nd fter insemintion. Ewes were inseminted by cervicl or intruterine lproscopic AI with F-EJAC, F-EP nd F-EPSP spermtozo (with motility 70%). 164 ewes were inseminted cerviclly (F-EJAC=77, F-EP=41 nd F-EPSP=46) nd 139 lproscopiclly (F-EJAC=40, F-EP=46 nd F-EPSP=53). 5

6 Prior to cervicl insemintion, ll tretments were diluted to stock solution of spermtozo/ml with UHT milk. Cervicl insemintion pipettes were loded with 0.2mL of semen with 0.2mL cushion of ir either side, giving cervicl insemintion dose of spermtozo/ewe. Ewes were cerviclly inseminted to industry stndrds. Ewes were prepred for lproscopic insemintion with intrmusculr injections of Ketmil (150mg; Troy Ilium, Glendenning, Austrli) nd ACP 2 (cetylpromzine, 2mg; Delvet, Seven Hills, Austrli), followed by subcutneous injection of locl nesthetic (2mL of 2% Lignocine; Mvlb, Logn City, Austrli). Prior to lproscopic insemintion, stock solutions were diluted 1:1 with UHT milk to give concentrtion of spermtozo/ml. Lproscopic insemintion pipettes were loded with 0.05mL of semen with 0.2mL cushion of ir either side, giving lproscopic insemintion dose of spermtozo/horn/ewe. Ewes were lproscopiclly inseminted by experienced professionls to industry stndrds. 60 dys fter insemintion, ewes were subjected to ultrsound in order to determine pregnncy sttus. After lmbing, ewes were ssessed for mmmry glnd development nd suckling to determine the number of ewes which hd lmbed nd foetl loss s per Evns nd Mxwell (1987) Sttisticl Anlyses Sttisticl nlyses were crried out using GENSTAT (15 th Edition; VSN Interntionl, Hemel Hempsted, UK). Experiment 1 ws ssessed using liner mixed model nd experiment 2 using generlised liner mixed model, both ccounting for fixed nd rndom effects. Trnsformtions were used to ttin dt normlity where required. Mens re reported with ± stndrd error of the men. Results Experiment 1: The effect of seminl plsm on the in vitro function of epididyml spermtozo, with nd without subsequent cryopreservtion Motility prmeters The totl percentge of fresh motile spermtozo decresed significntly over time s expected (p<0.001), but tretment hd no significnt effect. F-EJAC hd significntly higher progressive motility thn F-EP nd F-EPSP tretments t ll time points (p<0.001; verge 67.7% ± 3.5%; 45.8% ± 5.2%; 42.8% ± 6.1%; figure 1). The interction of time nd tretment significntly influenced verge pth velocity (p<0.05; figure 1b), with both F-EP nd F-EPSP hving lower verge pth velocity (verge 120.8μm/s ± 6μm/s; 122.5μm/s ± 5.5μm/s) thn the F-EJAC tretment (verge 159.2μm/s ± 7μm/s) t 0 nd 3 hours. Similr results were obtined for both stright line (p<0.05) nd curviliner velocity (p=0.05). Amplitude of lterl hed displcement ws significntly 6

7 lower in the F-EJAC tretment (verge 7μm ± 0.3μm) versus F-EP nd F-EPSP (verge 8.1μm ± 0.4μm; 7.9μm ± 0.4μm) overll (p<0.001), with ll tretments decresing in displcement over time (p<0.05). F-EJAC demonstrted significntly higher bet cross frequency thn both F-EP nd F- EPSP t ll time points (p<0.001; verge 37.6Hz ± 1.2Hz; 33Hz ± 1.2Hz; 33.3Hz ± 1.2Hz). F-EJAC similrly hd greter linerity thn both F-EP nd F-EPSP cross the 6 hour incubtion (p<0.001; verge 87.6% ± 1.2%; 79% ± 2.1%; 77.8% ± 2.5%). Strightness yielded similr results, with F- EJAC hving significntly higher strightness on verge (p<0.001) nd ll tretments incresing in strightness over time (p=0.005). There ws smll but significnt difference in motility (p<0.001) between C-EJAC (92.7% ± 1.3%) nd both C-EP nd C-EPSP (85% ± 2.7%; 85.6% ± 2.6%) in the pre-freeze ssessment. Nevertheless, during the 6 hour post thw incubtion, ll three cryopreserved tretments hd significntly different motilities (p<0.001; verge C-EJAC 74.9% ± 1.7%;C-EP 20.4% ± 4%; C- EPSP 31.9% ± 4.9%; figure 3). Progressive motility ws significntly ffected by the interction between tretment nd time (p<0.05; figure 3b), however during the pre-freeze nd post thw ssessments, overll significnt differences only existed between C-EJAC (verge 42.4% ± 2.8%) nd both cryopreserved epididyml tretments (verge C-EP 9.3% ± 2.6%; C-EPSP 11.7% ± 3.2%). Overll, C-EJAC hd significntly higher verge pth velocity (verge 107.7μm/s ± 7μm/s ; figure 3c) thn both C-EP nd C-EPSP (verge 62.1μm/s ± 5.2μm/s; 67.3μm/s ± 6.3μm/s) over the prefreeze ssessment nd post thw incubtion (p<0.001), with similr results for curviliner velocity (p<0.001). The interction between tretment nd time significntly ffected stright line velocity with C-EJAC moving significntly fster thn epididyml tretments t ll time points (p<0.05; verge C-EJAC 91μm/s ± 6μm/s; C-EP 46.7μm/s ± 5.1μm/s; C-EPSP 49.5μm/s ± 5.5μm/s). C-EJAC hd significntly higher mplitude of lterl hed displcement thn C-EP nd C-EPSP overll (p<0.001; verge C-EJAC 6.6μm ± 0.4μm; C-EP 4.9μm ± 0.4μm; C-EPSP 5.1μm ± 0.4μm). While ll tretments were similr in the pre-freeze ssessment, C-EJAC hd significntly higher bet cross frequency thn epididyml tretments post thw (p<0.05; verge C-EJAC 37.8Hz ± 0.6Hz; C-EP 33.8Hz ± 1.3Hz; C-EPSP 32.8Hz ± 0.7Hz; figure 3d). C-EJAC hd significntly higher linerity thn epididyml tretments post thw (p<0.05; verge C-EJAC 54.4% ± 1.8%; C-EP 49.6% ± 2.7%; C- EPSP 49.5% ± 2.3%) with linerity incresing significntly over time (p<0.001). Strightness mesures yielded similr results, with C-EJAC (verge 85.1% ± 1.1%) performing significntly better thn C-EP nd C-EPSP (verge 80.4% ± 2.8%; 79.1% ± 2.5%) over the post thw ssessment (p<0.05) nd strightness incresing significntly (p<0.001) over time

8 Cervicl migrtion test Time hd significnt impct on the number of fresh spermtozo which progressed to 1cm (p<0.001). Tretment ws lso significnt (p<0.05; figure 2), with the F-EPSP tretment hving significntly lrger popultion of spermtozo t 1cm thn the F-EP tretment t 0 hours (F-EPSP 37.4 ± 9.4; F-EP 14.5 ± 5.3) nd 3 hours (F-EPSP 24.5 ± 5.4; F-EP 9.6 ± 3.2). In ddition, the vngurd spermtozoon from the F-EPSP tretment penetrted significntly further through cervicl mucus thn tht of the F-EP tretment t 0 hours (p<0.05; F-EPSP 2.8cm ± 0.3cm; F-EP 1.9cm ± 0.3cm; figure 2b). Time significntly reduced the vngurd distnce of both F-EJAC nd F-EPSP (p<0.001), but not F-EP. Contrry to the results from fresh spermtozo, the interction between tretment nd time significntly ffected the number of cryopreserved spermtozo which reched 1cm (p<0.05; figure 4). Across the pre-freeze ssessment nd 6 hour incubtion, C-EJAC nd C-EPSP tretments hd similr results (verge 9.6 ± 3; 9.9 ± 3.6), nd both hd significntly greter numbers of spermtozo thn the C-EP tretment t 1cm (verge 3.4 ± 1.3). Vngurd distnce ws significntly influenced by the interction of tretment nd time (p<0.05), with C-EJAC nd C-EPSP penetrting significntly further through cervicl mucus thn C-EP t 0 hours (C-EJAC 2.25cm ± 0.2cm; C-EP 1.1cm ± 0.1cm; C-EPSP 1.6cm ± 0.1cm) nd 3 hours (C-EJAC 2cm ± 0.2cm; C-EP 1.4cm ± 0.2cm; C-EPSP 2cm ± 0.3cm ; figure 4b). Correltion between motility nd mucus penetrtion There were very wek positive correltions (R 2 < 0.1) between motility prmeters of totl motility, progressive motility nd verge pth velocity nd mucus penetrtion vribles of number of spermtozo t 1cm nd vngurd distnce for fresh nd cryopreserved tretments. Experiment 2: The effect of seminl plsm on the in vivo fertility of fresh epididyml spermtozo Results for pregnncy rte, lmbing rte nd foetl loss re shown in Tble I. The interction between tretment nd method of insemintion significntly ffected pregnncy t 60 dys (p=0.05). F-EP hd significntly poorer fertility when inseminted cerviclly (7.3%) versus lproscopiclly (50.0%) nd compred to both F-EJAC nd F-EPSP tretments (20.8%; 37.0%) when ll were inseminted cerviclly. There were no significnt differences between tretments inseminted lproscopiclly. There were no significnt differences between pregnncy rtes nd lmbing rtes. Only single ewe (F-EP, lproscopiclly inseminted) borted her foetus between ultrsound nd lmbing. 8

9 Discussion This study hs shown tht while seminl plsm does not lter the overll motility chrcteristics of epididyml spermtozo, it does improve their bility to penetrte cervicl mucus nd trverse the ovine cervix. While rm seminl plsm hs been shown to hve beneficil effects in vitro, this is the first known report of beneficil effect of seminl plsm on the trnsport nd survivl of epididyml rm spermtozo fter cervicl insemintion in n ovine model. Furthermore, this study hs demonstrted tht seminl plsm ws ble to improve in vitro mucus penetrtion in the bsence of improvements in motility. This my suggest tht the bility of spermtozo to penetrte cervicl mucus is not linked to motility, but rther n unknown trit conferred by exposure to seminl plsm. These results re encourging, s they hve helped to estblish the importnce of seminl plsm in different elements of cervicl trnsit of spermtozo, lending support to its ppliction in dvnced reproductive technologies. Unexpectedly, the mjority of motility prmeters of both fresh nd cryopreserved epididyml spermtozo did not pper to improve with the ddition of seminl plsm, with epididyml spermtozo generlly yielding poorer results thn ejculted spermtozo regrdless of exposure. These results gree with study by Dott et l. (1979), who found tht the ddition of undiluted seminl plsm to rm epididyml spermtozo resulted in short lived increse, followed by significnt decline in motility by 3 hours fter exposure. Furthermore, Dott et l. (1979) found tht even exposing epididyml spermtozo to 30% seminl plsm for just 15 minutes cused cler decline in motility, hypothesising tht while seminl plsm is initilly stimultory, it is ultimtely detrimentl to motility. Similrly, Heise et l. (2010) found tht seminl plsm stimulted equine epididyml spermtozo progressive motility to the sme level of ejculted spermtozo when immeditely ssessed. Yet fter freezing, supplemented epididyml spermtozo hd progressive motility equivlent to unsupplemented epididyml spermtozo nd fr lower thn tht of ejculted spermtozo. Overll, these results nd those of the current study lend support to the ide tht exposing epididyml rm spermtozo to undiluted seminl plsm hs no long term benefits for motility. While the poor progressive motility of the epididyml tretments in this study my be due to extended cold storge prior to collection, lck of positive response my lso be explined by the different rections of ejculted nd epididyml spermtozo to seminl plsm. It hs been demonstrted tht post thw ddition of seminl plsm to cryopreserved ejculted rm spermtozo my increse motility (El-Hjj Ghoui et l., 2007; Bernrdini et l., 2011). In opposition, when Thuwnut nd Chtdrong (2009) supplemented feline epididyml spermtozo post thw, they found tht seminl plsm significntly decresed motility compred to control supplemented with Tris buffer. This lck of positive effect of seminl plsm could possibly be due to the inbility of 9

10 epididyml spermtozo to bind motility driving seminl plsm proteins. While not protein ffecting motility, the bovine BSP 3 protein is produced in the epididymis, but cn only be bound by ejculted nd not epididyml spermtozo (Souz et l., 2011). This suggests tht the interction between spermtozo nd seminl plsm my be more complex thn simply being in ech other s presence nd tht optiml sperm function is possibly combined result of epididyml mturtion nd ejcultion. Overll, the bility of epididyml spermtozo to penetrte cervicl mucus ws significntly improved with exposure to seminl plsm, supporting the hypothesis tht seminl plsm my id the pssge of both fresh nd cryopreserved spermtozo through cervicl mucus. In some cses, epididyml spermtozo exposed to seminl plsm outperformed ejculted spermtozo, which is likely to be due to individul vritions in mle fertility. These results gree with the work of Arngsmy et l. (2005), who reported tht epididyml bufflo spermtozo exposed to isolted heprin nd geltin binding seminl plsm proteins progressed significntly further in bufflo cervicl mucus thn the unexposed control. Similr results were presented by Mxwell et l. (1999), with cryopreserved ejculted rm spermtozo showing significnt positive response in cervicl mucus penetrtion to post thw supplementtion of 30% v/v seminl plsm in DBPS, compred to DPBS supplemented control. The interction between seminl plsm nd cervicl mucus remins somewht of mystery nd to dte, β defensin 126, seminl plsm glycoprotein which fcilittes the penetrtion of cervicl mucus, is the only studied exmple of biochemicl interction between the two (Tollner et l., 2008). It ws demonstrted tht while the ddition of β defensin 126 to wshed mcque spermtozo restored cervicl mucus penetrtion, dding ll seminl plsm proteins resulted in slight inhibition of mucus penetrtion. While the results of this study re not conclusive nd further investigtion into this complex reltionship is required, this evidence does encourge the ide tht cervicl trnsit of spermtozo is driven nd hevily influenced by the proteomic components of seminl plsm. None of the mesured motility vribles were significntly correlted to the bility of fresh or cryopreserved spermtozo to penetrte nturl cervicl mucus. These results conflict with the findings of severl previous studies in humns (Keel nd Webster, 1988; Ford et l., 1992) nd sheep (Suttiyotin et l., 1995; Robyo et l., 2008; Mrtínez-Rodríguez et l., 2012), which found tht prmeters including totl motility, progressive motility, mplitude of lterl hed displcement nd verge nd curviliner velocity were significntly positively correlted to vrious mesures of mucus penetrtion. The comprison between the current results nd these previous studies rises the question of wht other fctors re possibly influencing the interction between spermtozo nd cervicl mucus. While seminl plsm filed to gretly improve motility prmeters, it significntly improved 10

11 the bility of exposed epididyml spermtozo to pss through cervicl mucus, highlighting its potentil importnce in ssisting spermtozo to successfully trverse the ovine cervix. As hypothesised, when inseminted cerviclly, epididyml spermtozo hd miniml fertility compred to both ejculted spermtozo nd epididyml spermtozo exposed to seminl plsm. Furthermore, when inseminted lproscopiclly, ll three tretments yielded reltively high, equivlent pregnncy rtes t 60 dys. Given the negligible bortion rtes, it ws concluded tht there ws no significnt influence of tretment on foetl loss. The below verge pregnncy rtes for the ejculted tretment re believed to be due to poor qulity ejcultes from the rms used for collection. Nevertheless, these results suggest tht epididyml spermtozo struggled to overcome the cervicl brrier, nd were only ble to do so effectively following exposure to seminl plsm. In similr study using ejculted rm spermtozo, Mxwell et l. (1999) demonstrted tht while frozen thwed rm spermtozo hd improved in vivo fertilistion cpcity when resuspended in seminl plsm nd inseminted cerviclly, there ws no effect on pregnncy rtes of lproscopiclly inseminted ewes. A previous study of lproscopic insemintion with unsupplemented, cryopreserved epididyml rm spermtozo yielded pregnncy rtes s high s 87% (Ehling et l., 2006), supporting the current finding of comprbly high epididyml fertility when inseminted directly into the uterus. The use of epididyml spermtozo rther thn wshed, ejculted spermtozo in this study hs helped to confirm the beneficil effect of seminl plsm on cervicl trnsit of spermtozo. The consistency in these results gives substnce to the ide tht seminl plsm plys key role in successful migrtion through the femle reproductive trct to rech the end gol of fertilistion. Fctors tht limit the extent to which these results cn be generlised include vrition between mles nd the possibility of breed bsed differences. Innte vritions exist in the fertility of rms, with rm seminl plsm contining proteins of both beneficil nd detrimentl ntures nd the mount of ech correlted to the fertility of the individul (Yue et l., 2009). While severl replictes ccounted for vrition in the qulity of epididyml collections, different mle ws used for the ejculted nd epididyml tretments. Idelly, ejcultes nd seminl plsm would be collected from rm which is then culled for n epididyml collection, but this ws not logisticlly possible in this study. In order to ensure sttisticl vlidity nd resonble relibility, seminl plsm ws insted individully pooled from severl ejcultes of ech rm nd pplied to n epididyml collection from single rm. The second limiting fctor in this study is the influence of breed, which hs been shown to impct both in vitro mucus penetrtion (Richrdson et l., 2011) nd pregnncy rtes following cervicl insemintion (Donovn et l., 2004). As this study used 100% purebred Austrlin merinos, the effects seen here my not be implicit cross ll sheep breeds. 11

12 This study hs shown tht exposure to seminl plsm is vitl for norml migrtion of spermtozo through the ovine cervix. Furthermore, it hs demonstrted tht in vitro mesures of sperm function, such s motility, my not give n ccurte representtion of in vivo outcomes. Most importntly, this study hs shown tht seminl plsm significntly impcts both in vitro mucus penetrtion nd in vivo fertilising bility following cervicl insemintion, supporting the ide tht key role of seminl plsm is ssisting spermtozo during the initil stges of trnsport through the femle reproductive trct. Judging from the results of the current study, continued reserch into how seminl plsm supports successful cervicl trnsit of spermtozo my be the key to improving cervicl insemintion success rtes using both fresh nd cryopreserved rm semen. Acknowledgements We would like to thnk the stff of Arthursleigh for their ssistnce nd knowledge nd Roslyn Bthgte, Andrew Souter, Byron Biffin, Jessie Mddison, Ethn Mooney, Dnielle Johinke nd Cssndr Sturt for the time nd effort they put into the field tril. This study ws supported by the NSW Stud Merino Breeders Assocition Trust nd Tylor Pini ws supported by scholrship from the Austrlin Wool Eduction Trust

13 Figures ) Progressive motility (%)! Time (hrs)! b) Averge pth velocity (μm/s)! Time (hrs)! Fig 1. ) Progressive motility nd b) verge pth velocity of F-EJAC ( ), F-EP ( ) nd F-EPSP ( ) tretments over 6 hour incubtion period. Vlues re mens ± SEM. Within time points, denotes significnt differences (p<0.05)

14 401 ) No. of spermtozo t 1cm! b b b Time (hrs)! b) Distnce trvelled by vngurd spermtozoon (cm)! b b b b Time (hrs)! Fig. 2. ) Men number of spermtozo trvelling 1cm through nturl cervicl mucus nd b) distnce (cm) of the furthest spermtozoon fter 1 hour incubtion for F-EJAC (blck), F-EP (white) nd F-EPSP (grey) tretments over 6 hour ssessment. Vlues re mens ± SEM. Within time points, different letters denote significnt differences (p<0.05). 14

15 408 ) Totl motility (%)! PF Time (hrs)! 409 b) 60 Progressive motility (%)! PF Time (hrs)!

16 Averge pth velocity (μm/s)! c) 0 PF Time (hrs)! d) 45 Bet cross frequency (Hz)! PF Time (hrs)! Fig. 3. ) Totl motility b) progressive motility c) verge pth velocity d) bet cross frequency of C- EJAC ( ), C-EP ( ) nd C-EPSP ( ) tretments over pre-freeze nd 6 hour incubtion period. Vlues re mens ± SEM. Within time points, denotes significnt differences (p<0.05)

17 420 ) b No. of spermtozo t 1cm! b 0 PF Time (hrs)! 421 b) 3 Distnce trvelled by vngurd spermtozoon (cm)! b c b b b 0 PF Time (hrs)! Fig. 4. ) Men number of spermtozo trvelling 1cm through nturl cervicl mucus nd b) distnce (cm) of the furthest spermtozoon fter 1 hour incubtion for C-EJAC (blck), C-EP (white) nd C-EPSP (grey) tretments over the pre-freeze ssessment nd 6 hour post thw ssessment. Vlues re mens ± SEM. Within time points, different letters denote significnt differences (p<0.05). 17

18 Tble I. Pregnncy nd lmbing rtes nd foetl loss fter cervicl nd lproscopic intruterine insemintion of synchronised mture merino ewes with fresh ejculted rm spermtozo (F-EJAC), epididyml rm spermtozo (F-EP) nd epididyml rm spermtozo exposed to rm seminl plsm (F-EPSP) Tretment Insemintion method No. ewes inseminted No. ewes pregnnt t dy 60 (%) No. ewes lmbed (%) Foetl loss (%) F-EJAC F-EP F-EPSP Cervicl (20.8) 16 (20.8) 0 Lproscopic (42.5) 17 (42.5) 0 Cervicl 41 3 (7.3) b 3 (7.3) b 0 Lproscopic (50) 22 (47.8) 2.2 Cervicl (37) 17 (37) 0 Lproscopic (58.5) 31 (58.5) Pregnncy rtes determined by ultrsound t dy 60 fter insemintion Lmbing rtes determined by mmmry glnd ssessment fter the expected lmbing dte s per Evns nd Mxwell (1987),b Within insemintion method, different superscripts denote significnt differences (p<0.05)

19 References Arngsmy, A., Singh, L. P., Ahmed, N., Ansri, M. R. nd Rm, G. C. (2005). Isoltion nd chrcteriztion of heprin nd geltin binding bufflo seminl plsm proteins nd their effect on cud epididyml spermtozo. Anim. Reprod. Sci. 90, Bernrdini, A., Hozbor, F., Snchez, E., Fornés, M. W., Alberio, R. H. nd Cesri, A. (2011). Conserved rm seminl plsm proteins bind to the sperm membrne nd repir cryopreservtion dmge. Theriogenology. 76, Dcheux, J.-L. (1980). An in vitro perfusion technique to study epididyml secretion. IRCS Med. Sci. 8, 137 Donovn, A., Hnrhn, J. P., Kummen, E., Duffy, P. nd Bolnd, M. P. (2004). Fertility in the ewe following cervicl insemintion with fresh or frozen thwed semen t nturl or synchronised oestrus. Anim. Reprod. Sci. 84, Dott, H. M., Hrrison, R. A. P. nd Foster, G. C. A. (1979). The mintennce of motility nd the surfce properties of epididyml spermtozo from bull, rbbit nd rm in homologous seminl nd epididyml plsm. J. Reprod. Fertil. 55, Drurt, X. (2012). Sperm Interction with the Femle Reproductive Trct. Reprod. Domest. Anim. 47, Ehling, C., Rth, D., Struckmnn, C., Frenzel, A., Schindler, L. nd Niemnn, H. (2006). Utiliztion of frozen-thwed epididyml rm semen to preserve genetic diversity in Scrpie susceptible sheep breeds. Theriogenology. 66, El-Hjj Ghoui, R., Gilln, L., Thomson, P. C., Evns, G. nd Mxwell, W. M. C. (2007). Effect of seminl plsm frctions from entire nd vsectomized rms on the motility chrcteristics, membrne sttus, nd in vitro fertility of rm spermtozo. J. Androl. 28, Evns, G. nd Mxwell, W. M. C. (1987). 'Slmon's rtificil insemintion of sheep nd gots.' (Butterworths: Sydney.) Ford, W. C. L., Ponting, F. A., McLughlin, E. A., Rees, J. M. nd Hull, M. G. R. (1992). Controlling the swimming speed of humn sperm by vrying the incubtion temperture nd its effect on cervicl mucus penetrtion. Int. J. Androl. 15, Fournier-Delpech, S., Cols, G., Courot, M. nd Ortvnt, R. (1977). Observtions on the motility nd fertilizing bility of rm epididyml spermtozo. Ann. Biol. Anim., Biochim., Biophys. 17, Heise, A., Kähn, W., Volkmnn, D. H., Thompson, P. N. nd Gerber, D. (2010). Influence of seminl plsm on fertility of fresh nd frozen-thwed stllion epididyml spermtozo. Anim. Reprod. Sci. 118, Hori, T., Hgiud, K., Endo, S., Hym, A., Kwkmi, E. nd Tsutsui, T. (2005). Unilterl intruterine insemintion with cryopreserved cudl epididyml sperm recovered from refrigerted cnine epididymides. J. Vet. Med. Sci. 67,

20 Hori, T., Ichikw, M., Kwkmi, E. nd Tsutsui, T. (2004). Artificil insemintion of frozen epididyml sperm in begle dogs. J. Vet. Med. Sci. 66, Keel, B. A. nd Webster, B. W. (1988). Correltion of humn sperm motility chrcteristics with n in vitro cervicl mucus penetrtion test. Int. J. Gynecol. Obstet. 27, 477 Mrtínez-Rodríguez, C., Alvrez, M., Ordás, L., Chmorro, C. A., Mrtinez-Pstor, F., Anel, L. nd de Pz, P. (2012). Evlution of rm semen qulity using polycrylmide gel insted of cervicl mucus in the sperm penetrtion test. Theriogenology. 77, Mxwell, W. M. C., Evns, G., Mortimer, S. T., Gilln, L., Gelltly, E. S. nd McPhie, C. A. (1999). Norml fertility in ewes fter cervicl insemintion with frozen-thwed spermtozo supplemented with seminl plsm. Reprod., Fertil. Dev. 11, Monteiro, G. A., Pp, F. O., Zhn, F. S., Dellqu Jr, J. A., Melo, C. M., Mziero, R. R. D., Avnzi, B. R., Alvreng, M. A. nd Gusti, P. N. (2011). Cryopreservtion nd fertility of ejculted nd epididyml stllion sperm. Anim. Reprod. Sci. 127, Richrdson, L., Hnrhn, J. P., O Hr, L., Donovn, A., Fir, S., O'Sullivn, M., Crrington, S. D., Lonergn, P. nd Evns, A. C. O. (2011). Ewe breed differences in fertility fter cervicl AI with frozen thwed semen nd ssocited differences in sperm penetrtion nd physicochemicl properties of cervicl mucus. Anim. Reprod. Sci. 129, Robyo, I., Montenegro, V., Vldés, C. nd Cox, J. F. (2008). CASA ssessment of kinemtic prmeters of rm spermtozo nd their reltionship to migrtion efficiency in ruminnt cervicl mucus. Reprod. Domest. Anim. 43, Silber, S., Devroey, P., Tournye, H. nd Vn, S. A. (1995). Fertilizing cpcity of epididyml nd testiculr sperm using intrcytoplsmic sperm injection (ICSI). Reprod., Fertil. Dev. 7, Souz, C. E. A., Mour, A. A., Lim-Souz, A. C. nd Killin, G. J. (2011). Binding ptterns of seminl plsm plsm proteins on bovine epididyml nd ejculted sperm membrne. Arq. Brs. Med. Vet. Zootec. 63, Stout, M. A., Senz, J. R., Chenevert, J. F., Gentry, G. T., Bondioli, K. B. nd Godke, R. A. (2012). Cryopreserved ejculted nd epididyml sperm collected from the sme holstein bulls used for in vitro fertiliztion Reprod., Fertil. Dev. 25, Suttiyotin, P., Thwites, C. J., Snchez-Prtid, L. G. nd Setchell, B. P. (1995). Evlution of modified sperm penetrtion test in rm semen. Theriogenology. 44, Thuwnut, P. nd Chtdrong, K. (2009). Incubtion of post-thw epididyml ct spermtozo with seminl plsm. Reprod. Domest. Anim. 44, Tollner, T. L., Yudin, A. I., Treece, C. A., Overstreet, J. W. nd Cherr, G. N. (2008). Mcque sperm coting protein DEFB126 fcilittes sperm penetrtion of cervicl mucus. Hum. Reprod. 23,

21 Yue, W., Shi, L., Bi, Z., Ren, Y. nd Zho, Y. (2009). Sodium dodecyl sulfte (SDS)-polycrylmide gel electrophoresis of rm seminl plsm proteins nd their correltion with semen chrcteristics. Anim. Reprod. Sci. 116,